Discrepant In Vitro Cytotoxicity Results Can Be Explained by Method-Specific Differential Dependency on Pentose Phosphate Pathway-Associated Metabolism

Details

• Personal Author:
  Coyle JP ; Derk R ; Farcas M ; Kornberg T ; Rojanasakul LW ; Stueckle, Todd A.
• Description:
  Tetrazolium reduction and resazurin assays remain the mainstay of routine in vitro toxicity batteries. However, neglecting to verify the baseline assumption of interaction of test article with method employed can produce potentially erroneous characterization of cytotoxicity/cell proliferation for test articles. The current investigation aimed to demonstrate the results of several standard cytotoxicity and proliferation assays varies in dependence on contributions from the pentose phosphate pathway (PPP). Beas-2B cells were treated with graded concentrations of benzo[a]pyrene (B[alpha]P) for 24 and 48 hours prior to cytotoxicity/proliferation assessment with commonly used MTT, MTS, WST-1, and Alamar Blue assays. B[alpha]P caused increased transient enhancement of metabolism of each dye assessed. An inhibitor of the glucose-6-phosphate dehydrogenase, 6-aminonicotinamide (6-AN), dose-dependently reduced metabolism of each endpoint listed in order of magnitude: WST-1 > MTS > MTT > Alamar Blue without overt cytotoxicity, implicating differential sensitivity of each endpoint to the pentose phosphate pathway (PPP). Nanomolar doses of B[alpha]P caused increases in all endpoints to suggest an increased cell proliferation. However, B[alpha]P treatment increased phosphorylation of Chk-1(S345) and p-53(S15), suggesting reductions in proliferative capacity. Reduction in proliferation was confirmed upon assessment of cell doubling time, which was lengthened dose-dependently. These results demonstrate differential sensitivity of studied cytotoxicity assessments on the PPP, thus 1) decoupling "mitochondrial activity" as an interpretation of cellular formazan and Alamar Blue metabolism, and 2) demonstrating the implicit requirement for investigators to sufficiently confirm methods of cytotoxicity/proliferation in routine experimentation. The nuances of these method-specific extramitochondrial metabolism must be scrutinized to properly qualify specific endpoints employed. [Description provided by NIOSH]
• Subjects:
  Amides Biological Assay Coloring Agents Cytotoxins Energy Metabolism Environmental Exposure Enzymes Laboratories Occupational Health Phosphoric Acids Quality Control Safety Toxicology

  More +
• Keywords:
  Analytical Instruments Analytical Methods Analytical Processes Benzo(a)pyrene Bioassays Carboxylic Acids Cell Proliferation Cellular Effects Cytometric Analysis Cytotoxic Effects Cytotoxicity Dose Response Dyes Enzyme Inhibitors Exposure Assessment Exposure Levels Exposure Methods In Vitro Studies Laboratory Techniques Laboratory Testing Metabolic Study Metabolism Performance Tests Phosphates Quality Control Quality Standards Sensitivity Testing

  More +
• ISSN:
  1096-6080
• Document Type:
  Text
• Genre:
  Abstract or Summary
• Place as Subject:
  California ; OSHA Region 3 ; OSHA Region 9 ; West Virginia
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Division:
  HELD - Health Effects Laboratory Division
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  84
• Volume:
  174
• Issue:
  1
• NIOSHTIC Number:
  nn:20058873
• Citation:
  Toxicologist 2020 Mar; 174(1):84
• CAS Registry Number:
  6-Aminonicotinamide (CAS RN 329-89-5) ; Benzo[a]pyrene (CAS RN 50-32-8) ; Glucose 6-phosphate dehydrogenase (CAS RN 9001-40-5) ; Resazurin (CAS RN 550-82-3)
• Federal Fiscal Year:
  2020
• NORA Priority Area:
  Manufacturing
• Peer Reviewed:
  False
• Source Full Name:
  The Toxicologist. Society of Toxicology 59th Annual Meeting and ToxExpo, March 15-19, 2020, Anaheim, California
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:af827712436b07d3056078014c7f5549658bcaeada3b0bfe0c8beb2b52f49ef3a96ed7abbbe750e2a6f1bba6ed051680cd2c5c3e891ce782b265db7043d7b965
• Download URL:
  https://stacks.cdc.gov/view/cdc/223248/cdc_223248_DS1.pdf
• File Type:
  [PDF - 524.31 KB ]